Chemiluminescent methods and reagents for analyte detection

ABSTRACT

The present invention relates to chemiluminescent method and regent to detect analyte. One aspect of the current invention relates to using enzyme substrate that can be cleaved by target enzyme to release chemiluminescent compound giving light signal for the detection of varieties of target enzymes. Another aspect of the current invention relates to use chemiluminescent enzyme coupled with analyte binding molecules to detect specific analyte molecules in a homogenous phase.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the divisional application of U.S. application Ser.No. 12/287,916 filed on Oct. 15, 2008, which claims priority to U.S.Provisional Application No. 60/999,166 filed on Oct. 16, 2007. Theentire disclosure of the prior application is considered to be part ofthe disclosure of the instant application and is hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to chemiluminescent method and regent todetect analyte. One aspect of the current invention relates to usingenzyme substrate that can be cleaved by target enzyme to releasechemiluminescent compound giving light signal for the detection ofvarieties of target enzymes. Another aspect of the current inventionrelates to using chemiluminescent enzyme coupled with analyte bindingmolecules to detect specific analyte molecules in a homogenous phase.

BACKGROUND OF THE INVENTION

Advances in the biological, biomedical and pharmaceutical sciences haveaccelerated the pace of research and diagnostics to a level unparalleledto the past. With sequences of whole genome becoming available quicklyand successively, the assembly of large libraries of small molecules,the ability to move pharmaceutical development, clinical diagnostictests and basic research from a reductionist to a whole system approachquickly all demand assays that facilitate high throughput analyses.Chemiluminescence (sometimes “chemoluminescence”) is the emission oflight (luminescence) with limited emission of heat as the result of achemical reaction. Light-emitting systems have been known and isolatedfrom many luminescent organisms, including certain bacteria, protozoa,coelenterates, mollusks, fish, millipedes, flies, fungi, worms,crustaceans, and beetles. Those enzymes isolated from beetles,particularly the fireflies of the genera Photinus, Photuris and Luciolaand click beetles of genus Pyrophorus have found widespread use inreporter systems. In many of these organisms, enzymatically catalyzedoxidoreductions take place in which the free energy change is utilizedto excite a molecule to a high-energy state. When the excited moleculespontaneously returns to the ground state, visible light is emitted.This emitted light is called “bioluminescence” or “chemoluminescence”.Luminescent luciferase-based assays have been developed to monitor ormeasure kinase activity, P450 activity, and protease activity. Fireflyluciferase or click beetle luciferase catalyses the oxidation of fireflyluciferin in the presence of ATP, Mg²⁺ and molecular oxygen with theresultant production of light. This reaction has a quantum yield ofabout 0.88 and this light emitting property has led to its use inluminescent assays. There are also other types of luciferin that cantrigger luminescent reaction. Bacterial luciferin is a reducedriboflavin phosphate (FMNH2, pictured here), which is oxidized inassociation with a long-chain aldehyde, oxygen, and a bacterialluciferase. Dinoflagellate luciferin is derived from chlorophyll, andhas a very similar structure. In the genus Gonyaulax, at pH 8 themolecule is “protected” from the luciferase by a “luciferin-bindingprotein”, but when the pH lowers to around 6, the free luciferin reactsand light is produced. Vargulin is found in the ostracod (“seed shrimp”)Vargula, and is also used by the midshipman fish Porichthys. Here thereis a clear dietary link, with fish losing their ability to luminesceuntil they are fed with luciferin-bearing food. Coelenterazine is themost “popular” of the marine luciferins, found in a variety of phyla.This molecule can occur in luciferin-luciferase systems, and is famousfor being the light emitter of the photoprotein “aequorin”. Besidesenzyme-catalyzed chemoluminescence, small organic molecule basedchemiluminescence assays are also widely used for analyte detection. Themost important chemiluminescent compounds include luminol, acridiniumand 1,2-dioxetane.

SUMMARY OF THE INVENTION

The present invention relates to chemiluminescent reagent and methodsfor analyte detection. These reagents and methods disclosed in thepresent invention enable simple, rapid and sensitive detection of theanalyte.

One aspect of the current invention involves the use of thechemiluminescent compound-enzyme substrate conjugate to detect thepresence of target enzyme. The enzyme breaks the conjugate and releasesthe chemiluminescent compound. The chemiluminescent compound can emitdetectable light under suitable conditions and therefore indicate thepresence of certain target enzyme. The chemiluminescent compounds usedin the current invention are firefly luciferin or 1,2-dioxetane. Theenzyme can be detected include alpha-L-Arabinosidase,beta-Cellobiosidase, alpha-L-Fucosidase, beta-D-fucosidase,beta-L-Fucosidase, alpha-Galactosaminidase, beta-Galactosaminidase,alpha-Galactosidase, beta-Galactosidase, alpha-Glucosaminidase,beta-Glucosaminidase, alpha-Glucosidase, beta-Glucosidase,beta-Glucuronidase, beta-Lactosidase, alpha-Maltosidase,alpha-Mannosidase, beta-Mannosidase, beta-Xylosidase, neuraminidase,proline aminopeptidase, leukocyte esterase, alpha-L-fucosidase,glycylproline dipeptidyl aminopeptidase, beta-galactosaminidase,N-acetyl-beta-D-glucosaminidase, Salmonella esterase, beta-glucuronidaseand hydroxyproline aminopeptidase.

Another aspect of the present invention provides methods andcompositions for firefly luciferase based homogeneous enzyme channelingluminescent assays for analyte detection.

The disclosed invention utilizes the enzyme channeling effect to detectthe analyte therefore enable simple, rapid and sensitive detection ofthe analyte without any separation steps. In some embodiments, the twoenzymes utilized for enzyme channeling are firefly luciferin producingenzyme and firefly luciferase. In other embodiments, those two enzymeutilized are ATP producing enzyme and firefly luciferase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the chemical structure of N-acetylneuraminic acid.

FIG. 2 is the chemical structure of firefly luciferin.

FIG. 3 is a chemical structure of an N-acetylneuraminic acid-fireflyluciferin conjugate.

FIG. 4 provides a schematic drawing showing the principle for influenzaneuraminidase detection as a means for the detection of influenza virususing the N-acetylneuraminic acid-firefly luciferin conjugate.

FIG. 5 is chemical structure of coelenterazine.

FIG. 6 provides coelenterazine (or its derivatives)—N-acetylneuraminicacid conjugate as substrate for sialidase detection, where R1, R2, R3,R4=H or alkyl group such as methyl group.

FIG. 7 provides an example of 1,2-dioxetane-sialic acid conjugate assubstrate for sialidase detection used in bacterial vaginosis test orChagas disease.

FIG. 8 provides additional examples of 1,2-dioxetane-sialic acidconjugates and derivatized forms of the conjugates as substrates forsialidase detection used in bacterial vaginosis test or Chagas diseasetest.

FIG. 9 shows L-prolyl-6-amino firefly luciferin.

FIG. 10 shows an example of L-prolyl-amino dioxetane.

FIG. 11 shows examples of substrates suitable for LE activity detection.

FIG. 12 shows examples of chemiluminescent acridinium derivatives.

FIG. 13 shows examples of substrate for AFU activity detection.

FIG. 14 shows examples of substrate for GPDA assay.

FIG. 15 shows firefly dehydroluciferin.

FIG. 16 provides a schematic drawing showing the luminesentacetylcholine esterase inhibition test using acetyl-fireflydehydroluciferin as substrate.

FIG. 17 provides example of chemiluminescent beta-galactosaminidasesubstrates.

FIG. 18 provides example of chemiluminescent substrates for NAGase.

FIG. 19 provides example of chemiluminescent substrates for salmonellaesterase.

FIG. 20 provides example of chemiluminescent substrates forbeta-glucuronidase.

FIG. 21 provides example of chemiluminescent hydroxyprolineaminopeptidase substrates.

FIG. 22 provides an example of enzyme channeling based detection system.

DETAILED DESCRIPTION OF THE INVENTIONS

The conjugate of N-acetylneuraminic acid (FIG. 1) and firefly luciferin(FIG. 2) is substrate for neuraminidase. N-acetylneuraminic acid is alsocalled sialic acid. A number of organisms express neuraminidase that canhydrolyze the N-acetylneuraminic acid-firefly luciferin conjugate (FIG.3). For example, influenza virus, parainfluenza and certain bacterialspecies possess neuraminidases. One important aspect of the currentinvention is the use of N-acetylneuraminic acid-firefly luciferinconjugate for specific detection of a neuraminidase from a particularorganism.

According to one embodiment of the invention, in order to detect certainspecies of neuraminidase activity, the undesired interferingneuraminidase activity from other species is inhibited using specificpolyclonal or monoclonal antibodies. For example, for detection ofinfluenza viral neuraminidase, the non-specific neuraminidase activityfrom likely contaminating organisms in the sample such as bacterialspecies Streptococcus pneumoniae and Actinomyces viscosus are inhibitedusing antibodies specific for the neuraminidases from these sources.This approach is possible because neuraminidases from differentorganisms have distinct amino acid sequences, which permits thegeneration of species-specific, or sub-species-specific, neuraminidaseantibodies. For example, specific antibodies are commonly used todifferentiate neuraminidase types of influenza virus in neuraminidaseneutralization assays.

The procedure of making these antibodies is well to the skilled in theart. For example, recombinant neuraminidase protein can be produced inE. coli by cloning the complete or partial genomic (in the case ofbacterial neuraminidase) or cDNA (in the case of eukaryoticneuraminidase) sequences into a bacterial expression vector thatpreferably contains an affinity ligand, e.g., his-tag, which facilitatesthe purification of the recombinant protein. Bacterial clones expressingthe enzyme can be screened and selected in a chemiluminescence assayusing the N-neuraminic acid-firefly luciferin conjugate. If therecombinant enzyme is tagged with an affinity ligand, then appropriateaffinity column can be used to purify the enzyme. For example, nickelcoated agarose column can be used to purify his-tagged recombinantneuraminidase. The purified neuraminidase can be used to immunize ananimal, e.g., a rabbit, for production of polyclonal antibodies.Alternatively, the protein can be used to immunize mouse from which theB-lymphocytes can be used to generate hybridoma, which can be used forscreening a monoclonal antibody that specifically inhibits theneuraminidase.

One or more monoclonal and/or polyclonal antibodies can be used in anassay for inhibiting neuraminidase activities from one or morecontaminating sources. For example, polyclonal antibodies or anti-seraor monoclonal antibodies for Streptococcus pneumoniae, Actinomycesviscosus and parainfluenza neuraminidases can be used in an assay fordetecting influenza viral neuraminidase. It is understood that theamounts of each antibody or anti-serum used in an assay need to beoptimized so that the antibodies can maximally inhibit contaminatingneuraminidase activities but not the neuraminidase activity to bedetected.

This method can be generalized for the detection of specific enzyme. Forexample, if enzyme A and B use the same substrate, in order toselectively detect enzyme B in the presence of enzyme A, the antibodyagainst enzyme A activity but not against enzyme B activity can be addedto the assay based on the detection of enzyme activity to decrease theinterference from enzyme A.

Besides fire fly luciferin, coelenterates type luciferin can also beused to synthesize a glycoside with sialic acid or its derivatives viathe phenol group on coelenterates. This kind of glycosides is alsoappropriate for use in the detection of neuraminidase activity, using aprocedure similar to the detection of neuraminidase.

The principle for detecting neuraminidase activity using the conjugateis depicted in FIG. 4. The sialic acid-firefly luciferin conjugate is asubstrate for neuraminidase, which cleaves the substrate to give rise tofree firefly luciferin that is the substrate of luciferase, which ispresent in the detection mix. The conjugate itself is not a substratefor firefly luciferase. Therefore, the luciferase-catalyzedbiochemiluminescence is dependent on neuraminidase activity, which isprovided by the influenza virus or other organisms to be detected. Asdescribed above, specificity of the assay can be achieved through theuse of inhibiting antibodies or modification of the sialic acid moietyof the conjugate. The drawing shows the principle for neuraminidase(e.g. influenza neuraminidase) detection as a means for the detection ofinfluenza virus using the N-acetylneuraminic acid-firefly luciferinconjugate. However, the same principle works for the neuraminidase fromother species using the same substrate. Further more, the same principlealso works for other enzyme when suitable chemiluminescent substrate isused as long as the substrate can be cleaved by the target enzyme andthe cleavage product can emit light for detection.

The assay can be done in one step or two-step fashion. The two-stepassay separates the enzyme cleavage step with the chemiluminescent step.The key feature of one-step method is the combination of target enzyme(e.g. neuraminidase) reaction with the chemiluminescence reaction in asingle step. In this assay format, luciferin is detected as it is beingreleased through the action of target enzyme (e.g. neuraminidase). Thismethod is therefore referred to as real time detection of target enzyme(e.g. neuraminidase) or real time detection method. However, in the realtime enzyme detection method, the detection mix contains all necessarychemicals and appropriate buffer for target enzyme reaction, includingthe conjugate itself, and for luciferase-catalyzed chemiluminescencereaction except for luciferin.

In the current invention, the term chemiluminescence and bioluminescenceare used interchangeable. Luciferin and luciferase are not specificmolecules. They are generic terms for a substrate and its associatedenzyme (or protein) that catalyze a light-producing reaction. A varietyof species regulate their light production using different luciferasesin a variety of light-emitting reactions. Luciferins are a class ofsmall-molecule substrates, each being specific for its correspondingprotein enzyme luciferase. Luciferins are catalyzed in the presence ofthe enzyme luciferase to produce light.

In the current inventions described above and below, the term fireflyluciferase include both the native firefly luciferase extracted fromfirefly and those engineered firefly luciferase such as those generatedby mutation for better thermal stability or different optimum pH oremission wavelength. There are many engineered firefly luciferase thatcan be found in scientific publications and patents and many of them arecommercially available. Any firefly luciferase is suitable for thecurrent invention as long as it uses firefly luciferin or fireflyluciferin directives (e.g. 6-amino firefly luciferin) for luminescence.Since there are also other luciferases that utilize firefly luciferin toemit light, for example, the click beetle luciferase, these luciferasescan also be used to replace the firefly luciferase used in the currentinvention.

There are several general types of luciferins: Firefly luciferin is theluciferin found in fireflies. It is the substrate of firefly luciferase.Bacterial luciferin is a type of luciferin found in bacteria, some squidand fish. It consists of a long-chain aldehyde and a reduced riboflavinphosphate. Dinoflagellate luciferin is a chlorophyll derivative and isfound in dinoflagellates, which are often responsible for the phenomenonof nighttime ocean phosphorescence. A very similar type of luciferin isfound in some types of euphausiid shrimp. Another luciferin calledvargulin is found in certain deep-sea fish, specifically, in ostracodsand poricthys. It is an imidazolopyrazine. The fifth luciferin calledcoelenterazine is found in radiolarians, ctenophores, cnidarians, squid,copepods, chaetognaths, fish and shrimp. It is the light-emittingmolecule in the protein called aequorin. Yet another luciferin is calledlatia luciferin which can be found in sea latia neritoides.

The luciferins suitable in the current invention are firefly luciferinand coelenterazine (FIG. 5). The coelenterazine-sialic acid conjugatecan also be cleaved by sialidase and the released coelenterazine canproduce light signal under the presence of aequorin. FIG. 6 provides thestructures of a list of coelenterazine (or itsderivatives)—N-acetylneuraminic acid conjugates, which can be used assubstrates in a sialidase assay such as Flu test or bacterial vaginosistest described below.

The methods, reagents and kits described above can also be used fordetection of other sialidases of bacterial, viral, protozoa, andvertebrate (including human) origin besides sialidase from influenzavirus. Sialidase (also known as neuraminidase or acylneuraminylhydrolase) is a protein enzyme produced by many organisms such asbacteria, viruses, protozoa, and vertebrates including humans. Thisclass of enzymes catalyzes the hydrolysis of terminal sialic acids whichare alpha-ketosidically linked to glycoproteins, glycolipids, andpolysaccharides through an O-glycosidic bond. There are a large numberof biological functions ascribed to sialidase enzyme, includingcell-cell recognition and pathogenicity of some infections bysialidase-bearing microorganisms.

In bacteria, sialidase helps bacterial adhesion to tissues and generatesadditional nutritional sources. In the case of the influenza virus,sialidase is one of two surface glycoproteins and is considered to beimportant for both transporting the virus through mucin and for thebudding of virus progeny from the infected cells. In the parasiteTrypanosoma cruzi, a sialidase (also known as trans-sialidase) removessialic acids from the infected cells and decorates its own surface withthese sialic acids. In humans, sialidases are involved in proteindegradation, immune responses, and cell proliferation. Abnormalproduction of sialidases may lead to serious human diseases such assialidosis or increased Pseudomonas aeruginosa infection in cysticfibrosis patients.

Since sialidases are associated with many diseases, a chemiluminescentsubstrate (e.g., the N-acetylneuraminic acid-firefly luciferin conjugatedescribed above) of sialidase would be an excellent diagnostic orprognostic reagent for sialidase-related diseases. For instance,sialidase level is elevated in bacterial vaginosis. Measurement ofsialidase level in the vaginal samples such as vaginal fluids can beused to diagnose bacterial vaginosis. Therefore, the kits and reagentsand methods described above can also be used to diagnose bacterialvaginosis.

In addition to the luciferin-N-acetylneuraminic acid conjugate, thechemiluminescent dioxetane-sialic acid conjugates, or its variations,are also suitable for use in the diagnosis of bacterial vaginosis,Chagas disease, or other diseases and conditions where sialidase is anappropriate marker. The structures for some of the substrates are shownin FIG. 7 and FIG. 8. It is understood that variations of theseconjugates may (e.g. the dioxetane can be selected from the dioxetanedescribed in U.S. Pat. Nos. 7,081,352 and 6,555,698) also be appropriatefor detecting sialidase as the marker.

Substrates depicted in FIGS. 7 and 8 or similar substrates can besynthesized using protocols that are described in U.S. Pat. Nos.7,081,352 and 6,555,698 as well as in a publication (AnalyticalBiochemistry 2000; 280, 291-300). Hydroxyl groups at the 4′ and 7′positions of the sialic acid moiety is preferred for detection ofbacterial sialidase since the 4, 7 alkylated substrates are morespecific to viral sialidase.

Chagas' disease (also called American trypanosomiasis) is a humantropical parasitic disease, which occurs in the Americas, particularlyin South America. Its pathogenic agent is a flagellate protozoan namedTrypanosoma cruzi, which is transmitted to humans and other mammalsmostly by blood-sucking bugs of the subfamily Triatominae (FamilyReduviidae). The cell invasion form of T. cruzi, Trypomastigote,expresses high levels of trans-sialidase activity; therefore,measurement of sialidase level can be used for diagnosis of active T.cruzi infection and for monitoring disease or therapeutic progress.

The substrates described above can also be used for the diagnosis of T.cruzi infection and Chaga's disease. As in the bacterial vaginosisdiagnostic tests, either N-acetylneuraminic acid-luciferin orN-acetylneuraminic acid-dioxetane conjugate, including theirderivatives, can be used for detection of T. cruzi infection. T. cruziinfection can be diagnosed with an assay that uses eitherN-acetylneuraminic acid-luciferin or N-acetylneuraminic acid-dioxetaneconjugate. There are several different types of infection status: 1)acute infection, which is an acute phase of an infection, 2) chronicactive infection, which an infection with persistent and activeinfection, and 3) cleared or dormant infection, which is an infectionwithout active infection but with detectable antibodies specific for theprotozoa.

For diagnosis of acute and active or chronic infection, sialidaseactivity in plasma or serum is measured using the chemiluminescent assaydescribed in this invention. Elevated sialidase activity in plasma orserum indicates active T. cruzi infection. In an active infection assay,serum or plasma sample with appropriate dilution in a buffer (e.g., PBSbuffer) as determined with experiments is added to a detection mix asdescribed in Example 2. The detection mix is preferably lyophilized forlong-term storage. The output signal is then measured with aluminometer. Again, a cutoff value for diagnosis positive needs to beestablished by testing a large number of negative samples, e.g., 100 ormore of negative samples. Sensitivity of the assay can be determined bytesting a large number of positive samples, e.g., 100 or more ofpositive samples confirmed with another method such as polymerase chainreaction (PCR) method.

T. cruzi infection may be cleared by the host but still results indetectable antibodies, which can be detected with a neutralization assaythat uses the same detection mix as for an active infection test exceptthat the detection mix also contains small amounts of T. cruzisialidase. In the absence of specific antibodies in a serum or plasmasample, the T. cruzi sialidase in the detection mix generates adetectable light signal at certain level. In the presence of specificantibodies in a serum or plasma sample, a reduction of the signal can bedetected. Thus, a reduction of the signal indicates chronic, cleared ordormant infection. Again, a cutoff value needs to be established bytesting a large number of negative samples, e.g., 100 or more ofnegative samples. Sensitivity of the assay can be determined by testinga large number of positive samples, e.g., 100 or more of positivesamples confirmed with another method such as an ELISA test.

In order to block the activity of human endogenous sialidase in thesample, which may interfere the measurement of T. cruzi sialidaseactivity, suitable amount of antibody against human endogenoussialidase, which can block its catalytic activity, can be added to thereaction mix. However, this antibody should not block the activity ofthe sialidase of T. cruzi.

In periodontal disease caused by bacterial infection, it has been shownthat the presence of sialidase increases the colonization of harmfulbacteria. In cystic fibrosis patients, Pseudomonas aeruginosa infectionis one of the leading causes of death. Sialidase was shown to beinvolved in the disease progress. Sialidase is also related to theregulation of cell proliferation, the clearance of plasma proteins, andthe catabolism of gangliosides and glycoproteins. Therefore, the reagentand method used for bacterial vaginosis described in the currentinvention can also be used for the diagnosis for these conditions.

Elevated proline aminopeptidase activity in vaginal fluid has beenassociated with bacterial vaginosis. Thus, a proline aminopeptidaseassay can also be used to diagnose bacterial vaginosis. Prolineaminopeptidase (or called proline iminopeptidase) is a hydrolase thatcleaves the L-proline residues from the N-terminal position in peptides.A substrate that can be used in a proline aminopeptidase assay forbacterial vaginosis diagnosis is L-proline-6-amino firefly luciferinconjugate (L-prolyl-6-amino firefly luciferin, FIG. 9) or itsderivatives. In a proline aminopeptidase assay, the enzyme cleaves thesynthetic substrate (FIG. 9) and releases the free 6-amino fireflyluciferin, which can be quantitatively detected in a chemiluminescencereaction in the presence of firefly luciferase and ATP. It can be aone-step assay or two step assays in a fashion similar to thosedescribed in the influenza or bacterial vaginosis sialidase assay. Oneskilled in the art can readily transfer the reagent and method used inthe bacterial vaginosis sialidase assay for the proline aminopeptidaseassay by replacing the substrate.

In other embodiments, L-proline-1,2-dioxetane derivative conjugates,illustrated in FIG. 10 as an example, are used in a chemiluminescentproline aminopeptidase assay for detection of bacterial vaginosis.Instead of using 6-amino firefly luciferin as the chemiluminescentmoiety, these conjugates use amine containing dioxetane derivative (suchas those listed in U.S. Pat. No. 5,843,681) as the chemiluminescentmoiety, which is conjugated to the —COOH group of L-proline through apeptide bond as shown in FIG. 10 as an example. Synthesis of this groupof conjugates can be accomplished using peptide synthesis chemistry,which is well known to the skilled in the art.

Detection protocols for the chemiluminescent proline aminopeptidaseassay can be adopted from those described in Example 4 whenL-proline-luciferin is used as the substrate. The peptidase assayprotocol can also be adopted from well know resource (e.g. U.S. Pat. No.5,843,681) by the skilled in the art when L-proline-dioxetane is used asthe substrate. Again, a cutoff value needs to be established by testinga large number of negative samples, e.g., 100 or more of negativesamples. Sensitivity of the assay can be determined by testing a largenumber of positive samples, e.g., 100 or more of positive samples.

Elevated leukocyte esterase activity indicates the presence of whiteblood cells and other abnormalities associated with infection orinflammation. Leukocyte esterase test (LE test) is widely used to detecta leukocyte esterase, an enzyme released by white blood cells, whichsuggests the presence of leukocytes (white blood cells) in the sample,e.g. LE in the urine, which in turn probably indicates active urinarytract infection. LE tests are also used to screen for gonorrheainfection, colpitis, amniotic fluid infections, bacterial meningitis,and ascite or hydrothorax infection by testing leukocyte esteraseactivity in appropriate samples. Current invention involveschemiluminescent substrates and methods for leukocyte esterase test. Thesubstrates are carboxylic acid-firefly luciferin conjugates such asacetyl firefly luciferin. The —COOH group of the carboxylic acid arecoupled with the 6-OH group of the firefly luciferin to generate anester bond. Suitable carboxylic acids include, but are not limited to,alkyl substituted carboxylic acid such as formic acid, acetic acid,propionic acid, butyric acid and etc. as well as aromatic acid such asbenzoic acid. The esterase hydrolyzes the ester bond of the conjugate torelease free firefly luciferin, which becomes a substrate for luciferasein a biochemiluminescent reaction that generates a light signal.Alternatively, the carboxylic acid-1,2-dioxetane conjugates are alsosuitable substrates for chemiluminescent LE activity detection. Thesedioxetane conjugates can be structurally similar to those that aredepicted in FIGS. 7 and 8, where the sugar moiety (N-acetylneuraminicacid) is replaced with the carboxyl group, e.g. an acetyl group, to forman ester bond. In still certain embodiments, the substrate are thefirefly luciferin-alcohol conjugates, which is the ester formed bycoupling the —COOH group of firefly luciferin with the —OH of analcohol, resulting in, for example, firefly luciferin methyl ester orfirefly luciferin ethyl ester. The alcohol can be either alkyl alcoholsuch as methanol, glycerol or aromatic alcohol such as benzyl alcohol.The esterase hydrolyzes the ester bond of the conjugate to release freefirefly luciferin, which becomes a substrate for luciferase in abiochemiluminescent reaction that generates a light signal. Elevatedlight signal indicates high LE activity, which in turn is indicative ofan active infection. Three examples suitable for LE activity detectionis shown in FIG. 11, where n could be any integers between 0 and 3.Again, a cutoff value needs to be established by testing a large numberof negative samples, e.g., 100 or more of negative samples. Sensitivityof the assay can be determined by testing a large number of positivesamples, e.g., 100 or more of positive samples.

Hydrogen peroxide (H₂O₂) producing lactobacilli play an important rolein preventing vaginal infections by controlling the microbial flora invagina. Women colonized by lactobacilli have decreased acquisition ofvaginal infections because the H₂O₂ produced inhibits the growth ofpathogenic bacteria. A test that can quantitatively detect H₂O₂ in thevaginal sample can be used to assess the health of vaginalmicroenvironment and for the diagnosis of bacterial vaginosis and othervaginosis such as yeast infection. The test described in the currentinvention utilizes a chemiluminescent substrate that can generate alight signal when mixed with H₂O₂. Examples of suitable substratesinclude acridinium derivatives and luminol. Examples of some acridiniumderivatives are shown in FIG. 12. A variety of acridinium derivativesincluding amine-derivatized, carboxyl-derivatized and NHS esterderivatized acridinium are commercially available or can be found from awell-known reference. It is understood that other H₂O₂ dependentchemiluminescent chemicals, known or unknown, may be appropriate fordetecting hydrogen peroxide in vaginal fluid samples as an indicator forvaginal infection status so long as the chemiluminescent chemicalsenable a chemiluminescent reaction that is dependent on hydrogenperoxide. Elevated pH (e.g. pH=8˜11) or the presence of peroxidase (e.g.horse radish peroxidase) increase the release speed of active oxygenfrom H₂O₂. The released active oxygen causes the acridinium or luminolto undergo oxidation and emit light that can be detected with aluminometer. Therefore assay conditions such as elevated pH (e.g.pH=8˜11) or addition of peroxidase can increase the light signal in theH₂O₂ detection assay. The optimal peroxidase amount need to be added orthe optimal pH can be determined experimentally according to protocolsthat are well known to the skilled in the art. Similar to the bacterialvaginosis test using luciferin or dioxetane conjugates, the test kit fora hydrogen peroxide test may have to kit components, a samplepreparation buffer (e.g., 1 mL of 0.1 M NaHCO₃, pH 8.5) and a detectionmix. The detection mix solution (e.g., 1 mL 0.1M NaHCO₃, pH 8.5 and 0.1micromole of acridinium or luminol) can be used as it is or, preferably,is lyophilized for long-term storage. The detection protocol can be asfollows: vaginal fluid is collected with a vaginal swab, which is rinsedin the sample preparation buffer. The resulting sample is then added tothe detection mix and then subjected to detection using a luminometer.The light signal output is measured for a period of time, e.g., 10seconds to 3 minutes, and then integrated. The total signal output isthen used to determine the status of hydrogen peroxide in vaginal fluid.Again, a cutoff value can be established by testing a large number ofnegative clinical samples, e.g., 100 or more of negative samples.Sensitivity of the assay can be determined by testing a large number ofpositive clinical samples, e.g., 100 or more of positive samples. Thepositive samples are those from patients with vaginal infections(bacterial vaginosis or other microbial infection) that are confirmedwith other well-recognized methods.

It is within the scope of the present invention that a combination oftwo or more tests (e.g. hydrogen peroxide test, LE activity test,proline aminopeptidase test and sialidase test described above) may beused for diagnosis of a vaginal infection. For example, a hydrogenperoxide test and sialidase test can be used in combination to diagnosea vaginal infection other than bacterial vaginosis, where low sialidaseactivity (below the cutoff value of the bacterial vaginosis test) andhydrogen peroxide content (below the cutoff value for samples fromhealthy women) in vaginal fluid may indicate the presence ofnon-bacterial infection such as fungal infection.

The alpha-L-fucosidase (AFU) assay is for the determination of AFUactivity in patient serum or organ or other body fluid samples. AFU is alysosomal enzyme involved in the degradation of a diverse group ofnaturally occurring fucoglycoconjugates. Serum AFU activity isconsidered a useful marker of hepatocellular carcinoma (HCC). ElevatedAFU levels in serum are an early indication of HCC. Though measurementof serum fetoprotein (AFP) is a common practice for early detection ofHCC, use of AFP assay alone suffers from its low specificity andsensitivity, due to the fact that not all HCC secrete AFP. AFP levelsmay be normal in as many as 40% of patients with early HCC and 15-20% ofpatients with advanced HCC. Recent studies have clearly demonstratedthat measurements of both AFP and AFU can significantly increase thedetection specificity and sensitivity for HCC. AFU is reported to be amore sensitive marker, especially for detecting a small tumor size ofHCC. It has also been shown that abnormal AFU level exists in serumsamples from patients suffering from adult leukemia or ovariancarcinoma. In addition, AFU level is also high in patients sufferingfrom liver cirrhosis and chronic hepatitis. According to the presentinvention, AFU level in a sample is quantified using a syntheticsubstrate alpha-L-fucopyranoside-firefly luciferin conjugate, whosechemical structure is similar to FIG. 3, which depicts a conjugatebetween neuraminic acid and firefly luciferin. In thealpha-L-fucopyranoside-firefly luciferin conjugate, the neuraminic acidportion in FIG. 3 is replaced with alpha-L-fucopyranoside.

In a chemiluminescent AFU assay, alpha-L-fucopyranoside-fireflyluciferin conjugate is cleaved by alpha-L-fucosidase in a sample torelease free firefly luciferin, which can be quantified in achemiluminescence reaction in the presence of firefly luciferase, ATPand other appropriate conditions. It can be a one-step assay or twosteps assay as those described in the sialidase test for bacterialvaginosis diagnosis. The assay kit can be similar to those used forsialidase detection except the substrate is fireflyluciferin-alpha-L-fucopyranoside instead. The assay protocol can bereadily adopted from the bacterial vaginosis test described above by askilled in the art.

The 1,2-dioxetane derivative-alpha-L-fucopyranoside and coelenterazine(or its derivatives)—alpha-L-fucopyranoside are also suitable substratesfor chemiluminescent AFU activity detection. The structures of thedioxetane substrates can be similar to the structures depicted in FIGS.7 and 8 except the sugar moiety in the substrates isalpha-L-fucopyranoside. Two examples suitable for AFU activity detectionare shown in FIG. 13. A cutoff value for diagnosis can be established bytesting a large number of negative clinical samples, e.g., 100 or moreof negative samples. Sensitivity of the assay can be determined bytesting a large number of positive clinical samples, e.g., 100 or moreof positive samples. The positive samples are those from patients whoare confirmed positive for hepatocellular carcinoma (HCC) with otherwell-recognized methods.

Elevated glycylproline dipeptidyl aminopeptidase (GPDA) activity inblood and urine is associated with abnormality in liver, stomach,intestine and kidney. Elevated GPDA activity (especially the isoenzyme,GPDA-F) in serum has been identified as a reliable marker enzyme forhepatocellular carcinoma and other liver disease. The GPDA assay in thecurrent invention is based on the enzymatic cleavage of the syntheticsubstrate L-glycyl-L-prolyl-6-amino firefly luciferin, whose chemicalstructure is similar to that of L-prolyl-6-amino firefly luciferin,which is depicted in FIG. 9. In the L-glycyl-L-prolyl-6-amino fireflyluciferin conjugate, the peptide moiety is L-glycyl-L-proline. In theGPDA assay, the L-glycyl-L-prolyl-6-amino firefly luciferin conjugate iscleaved by GPDA to give rise to free luciferin, which is detected in afirefly luciferase catalyzed chemiluminescence reaction. It can be aone-step assay or two steps assay as described before. Reagent and kitformulation can be similar to those described in Examples 1 and 2 aswell.

The L-glycyl-L-prolyl-1,2-dioxetane conjugates, which are similar to thesubstrates depicted in above proline aminopeptidase assay(L-glycyl-L-prolyl is present in the substrate instead of L-prolylgroup), can also be used in chemiluminescent GPDA assay. In this assayformat, the cleaved dioxetane moiety in the conjugate gives rise to thelight signal that is dependent on GPDA activity in a sample. The assayprotocol and reagent formulation can be readily adopted from the prolineaminopeptidase assay for bacterial vaginosis detection as describedbefore. Two examples of the substrate suitable for GPDA assay are shownin the FIG. 14. Again, a cutoff value can be established by testing alarge number of negative clinical samples, e.g., 100 or more of negativesamples. Sensitivity of the assay can be determined by testing a largenumber of positive clinical samples, e.g., 100 or more of positivesamples. The positive samples are those from patients who are confirmedpositive for hepatocellular carcinoma (HCC) with other well-recognizedmethods.

The current invention also relates to methods and reagents for thedetection of organophosphorus agents, which include pesticides and nervegas. These organophosphorus chemical agents can bind and block esteraseenzymes, especially acetylcholine esterase (AChE), which is present inthe blood and at the neuromuscular junctions in the peripheral andcentral nervous systems. AChE regulates the level of theneurotransmitter acetylcholine (ACh) by degrading it. Theseorganophosphorus nerve agents inhibit AChE, resulting in excessiveaccumulation of Ach, which can fatally impair the nerve system of theindividuals who are exposed to it.

Firefly luciferin can be oxidized into a dehydrogenated form calledfirefly dehydroluciferin (FIG. 15), which is a potent inhibitor offirefly luciferase bioluminescence reaction with an IC₅₀ of 6 nM. Thus,acetyl-firefly dehydroluciferin can be used in a “positive” inhibitionassay for the detection of organophosphorous agents, as depicted in FIG.16. The reaction mix will contain four key reagents: theacetyl-dehydroluciferin, acetylcholine esterase, firefly luciferase andsmall amounts of firefly luciferin. In the absence of anorganophosphorous agent, acetylcholine esterase hydrolyzes the substrateand releases dehydroluciferin, which inhibits firefly luciferaseactivity and consequently reduces the light signal. Presence of anorganophosphorous agent, which inhibits the esterase, leads to areduction of dehydroluciferin resulting in an increase of signalintensity. Thus in this assay format, the signal intensity isproportional to the concentration of the organophosphorous agent.Firefly dehydroluciferin can be synthesized according to the proceduredescribed in the literature (E H White, F McCapra, G F Field—Journal ofthe American Chemical Society, 1963 v 85 p 337; title: The Structure andSynthesis of Firefly Luciferin) In brief, firefly luciferin is dissolvedin sodium hydroxide solution and the solution is boiled in air untilthin layer chromatography (TLC) shows the absence of firefly luciferin.This process may take several hours, e.g., 8 hours. The solution isacidified with concentrated hydrochloric acid and extracted with ethylacetate followed by silica gel column chromatography. Acetylization offirefly dehydroluciferin is performed by incubating the purified fireflydehydroluciferin with excessive amounts of acetic anhydride in pyridineat room temperature for 1 hour, followed by silica gel columnpurification to remove the unreacted firefly dehydroluciferin. Therecovered acetyl-firefly dehydroluciferin can be further purified usinga preparative HPLC to completely remove the firefly dehydroluciferin. Apreferred detection mix contains 0.2 M phosphate buffer, pH 7.5, 1 mg/mLATP, 15 mM magnesium sulfate, 4 mM calcium chloride, 0.1 nM fireflyluciferin, 10 nM acetyl-firefly dehydroluciferin, 100 microgram/mLacetylcholine esterase and 1 microgram/mL firefly luciferase. 200microliters of the reaction mix is lyophilized in a detection tube. Eachdetection tube is used for one sample testing. Other than the detectionmix, the test kit also needs a positive control (e.g., 1 microgram/mL oforganophospharus in 1× PBS buffer) and a negative control (e.g., 1× PBSbuffer). 200 microliters of sample is added to the lyophilized detectionmix, incubated for 5 minutes and then placed in the detection chamber ofa luminometer for detection. The light signal (RLU—relative light unit)is recorded. Background counts are determined using the negativecontrol. This substrate and method can also be used for other esteraseactivity detection such as LE activity detection described above.

The current invention also relates to novel chemiluminescent substratesthat can be used to detect beta-galactosaminidase activity, a biomarkerfor Candida albican, which is the most common pathogen that causes yeastinfection. The substrates that are suitable for chemiluminescentreaction based detection of beta-galactosaminidase are composed of twomoieties, the galactosamine moiety (e.g., acetyl-galactosamine) andchemiluminescent moiety (e.g., firefly luciferin or 1,2-dioxetane),which are linked together through an appropriate chemical bond that canbe cleaved by the beta-galactosaminidase. Examples of these substratesare shown in FIG. 17. In the presence of beta-galactosaminidase in asample, the conjugate is cleaved to release free chemiluminescentmoiety, firefly luciferin or 1,2-dioxetane, which becomes luminescentunder appropriate conditions. The emitted light can be detected with asimple luminometer. Formulations of the reagents and test kits anddetection procedures can be adopted from Examples 1 and 2 for fireflyluciferin containing substrates, and Examples 5 for dioxetane-containingsubstrates.

The released free firefly luciferin can be quantified by measuring thechemiluminescence in the presence of firefly luciferase and ATP usingthe protocol described above. Other substrates such as 1,2-dioxetanederivative-N-acetyl-beta-D-galactosaminide and coelenterazine (or itsderivatives)—N-acetyl-beta-D-galactosaminide (e.g. those same to thestructures in FIG. 6,7,8 except the sugar part isN-acetyl-beta-D-galactosaminide instead) are also suitable substratesfor chemiluminescent beta-galactosaminidase activity detection as longas there is a moiety that enables light emitting after cleavage with theenzyme.

Candida albicans produces both L-proline aminopeptidase andbeta-galactosaminidase enzymes whereas other yeast/bacterial speciesproduce only one or neither of the enzymes. Therefore the abovesubstrates can be used for candida albicans detection. Thesebeta-galactosaminidase substrates can be either used alone or, inpreferred embodiments, in combination with substrates for L-prolineaminopeptidase (e.g. those used for proline aminopeptidase BV assay). Asample with elevated activities for both enzymes is considered positivefor Candida albicans infection. A cutoff values for both enzymes can beestablished by testing a large number, e.g., 100, of negative samples.The sensitivity of the test can be evaluated by testing a large number,e.g., 100, of positive samples.

The composition of the beta-galactosaminidase test kit is similar tothose used in the sialidase test except the substrate for sialidase isreplaced with the substrate for beta-galactosaminidase. The amount ofthe substrate can be optimized experimentally for optimal detection. ThepH can also be optimized experimentally for optimal detection. Thecomposition of the L-proline aminopeptidase test kit and assay protocolcan be identical to or adopted from the proline aminopeptidase test usedin bacterial vaginosis detection.

The current invention also relates to novel chemiluminescentN-acetyl-beta-D-glucosaminidase (hereinafter simply referred to asNAGase) substrates that can be used to detect NAGase activity. TheNAGase can cleave the substrates and release theN-acetyl-beta-D-glucosamine and the chemiluminescent moiety such asfirefly luciferin or 1,2-dioxetane derivatives, which enableluminescence reaction when they are in their free form. Examples ofthese substrates are shown in the FIG. 18. The free firefly luciferincan be quantified using a biochemiluminescence assay in the presence offirefly luciferase/click beetle luciferase and ATP as described above.Other substrates such as 1,2-dioxetanederivative—N-acetyl-beta-D-glucosaminide and coelenterazine (or itsderivatives)—N-acetyl-beta-D-glucosaminide (such as those similar to thestructures depicted in FIG. 6,7,8 except the sugar part isN-acetyl-beta-D-glucosaminide instead) are also suitable for use inchemiluminescent NAGase activity detection as long as the releasedmoiety can be used to produce light signal.

NAGase is one of the enzymes in lysosomes distributed in the kidneytubular epithelium in large quantities, and participates indecomposition of glucoproteins and mucopolysaccharides. It is recognizedthat urinary NAGase activity increases in various renal diseases such asacute renal deficiency, glomerulonephritis, etc. or in post-operativekidney. It is also recognized that in the case of diabetes the amount ofNAGase increases not only in urine but also in serum. As an aid fordiagnosis and monitoring of various renal diseases and also as an indexin studies on renal toxicity of drugs, determination of NAGase activityhas attracted much attention both in clinical fields and in animalexperiments.

Substrates for use in determining NAGase activity currently usedinclude, for example, p-nitrophenyl-N-acetyl-beta-D-glucosaminide,4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide andm-cresolsulfonephthaleinyl-N-acetyl-beta-D-glucosaminide or thosedescribed in U.S. Pat. Nos. 5,274,086 and 5,030,721. However, they areall colorimetric based test, which has low sensitivity and requires longincubation time. One objective of the present invention is to providenovel compounds overcoming the problems mentioned above for determiningNAGase activity and methods of determination using these novelcompounds. The compounds and methods provided in the present inventionenable rapid and sensitive determination of NAGase activity.

A variety of sample sources are appropriate for use in determining theNAGase activities. Examples include culture fluids of microorganisms,plant extracts, body fluids, urine and tissues of animals and extractsthereof. If necessary, pretreatment of the sample may be performed. Inaddition, an oxidizing agent is added to minimize the effect ofreducible substances in the sample in some applications. Appropriatebuffers that can be used for the assay include, but are not limited to,phosphates, acetates, citrates, succinates, phthalates and etc. Theassay can be done in either the so-called end-point assay format, inwhich the enzyme reaction is once discontinued to perform thedetermination of the enzyme activity, or the rate-assay method, which isone of the most popular methods for determining enzyme activity in manycases. It can also be done by determining the time dependent RLU orintegrating the overall RLU in certain time window as described inprevious applications. Alternatively, it can be done in the real timeassay format, in which both reactions are performed simultaneously in asingle reaction tube. In the real time assay format, the signal(relative light units) can be recorded in a time-dependent manner tomeasure the kinetics of the reactions or the signal over a period oftime (e.g., 2 minutes) is integrated to measure an overall signalintensity.

The optimal pH for NAGase activity is 4.5-5.0. In the end point assayformat, the sample can be incubated with the substrate at this optimalpH for a certain period of time (e.g., 10 minutes) followed byadjustment of pH to an optimal value for the bioluminescence reaction(e.g. pH 7.8 for firefly luciferase) or for chemiluminescence (pH 10.5for dioxetane type substrate). In a specific example, thedioxetane-containing substrate is dissolved in 50 mM citrate buffer(pH=5.0) at an optimal concentration that is determined experimentally.1 mL of the substrate solution is mixed with 25 microliters of a samplesolution. The mixture is incubated at 25 degree C. for 5 minutes,followed by adjustment of the pH value to 10.5 using 0.2 M NaOHsolution. The light signal is measured using a luminometer. A standardcurve is established by 625 testing a number of samples containingvarying concentrations of NAGase activities. The NAGase activity in thesample solution is then determined by comparing the RLU from the samplewith the standard curve.

The Salmonella esterase catalyzes the hydrolysis of a variety of C6 toC16 fatty acid esters but does not hydrolyze peptide bonds. Presence ofthis esterase activity is indicative of Salmonella contamination in asample. The current invention also relates to novel chemiluminescent C6to C16 fatty acid ester esterase substrates that can be used to detectSalmonella. The Salmonella esterase can cleave the substrates andrelease the chemiluminescent moiety such as firefly luciferin or1,2-dioxetane derivatives, which can luminesce once cleaved from theirconjugates. The substrates useful for the current invention are fireflyluciferin fatty acid ester (C6 to C16 fatty acid) or 1,2-dioxetane fattyacid ester (C6 to C16 fatty acid). The —COOH group of the fatty acid arecoupled with the 6-OH group of the firefly luciferin or the —OH on the1,2-dioxetane to generate an ester bond. These esters are similar to thestructures in FIG. 6,7,8 and sialidase substrates in the BV test exceptthat the sugar part is replaced by the fatty acid group, e.g. ancaprylyl group, which form an ester bond. One preferred fatty acid iscaprylic acid. Examples of these substrates are shown in the FIG. 19. Inone embodiment, the dioxetane-containing substrate is dissolved in 1×PBS buffer (pH=7.5) at an optimal concentration that is determinedexperimentally. 0.5 mL of the substrate solution is mixed with 200microliters of a sample solution. The mixture is incubated at 25 degreeC. for 5 minutes, followed by adjustment of the pH value to 10.5 using0.2 M NaOH solution. The light signal is measured using a luminometer. Acutoff value can be established by testing a large number of negativesamples that contain no Salmonella esterase activities. Higher lightsignal indicate the presence of higher amount of Salmonellacontamination.

The current invention also relates to novel chemiluminescentbeta-glucuronidase substrates that can be used to detectbeta-glucuronidase activity. The beta-glucuronidase cleaves thesubstrates and release the beta-D-glucuronic acid and thechemiluminescent moiety such as firefly luciferin or 1,2-dioxetanederivatives, which can luminesce once cleaved from their conjugates andunder appropriate conditions. Examples of these substrates are shown inFIG. 20. The released free firefly luciferin can be quantified in abiochemiluminescence reaction that uses firefly luciferase and ATP.Protocols for this type of biochemiluminescent assays are describedabove (e.g., Examples 1 and 2). Other substrates such as 1,2-dioxetanederivative-beta-D-glucuronide and coelenterazine (or itsderivatives)—beta-D-glucuronide (e.g. those similar to the structures inFIG. 6,7,8 except that the sugar moiety is beta-D-glucuronide) are alsosuitable for use in detection of beta-glucuronidase activity in achemiluminescent assay as long as the released chemiluminescent moietycan be used to produce light signal. Quantitative detection ofbeta-glucuronidase activity has many applications. For example, manycarcinoma patients show elevated beta-glucuronidase (beta-G) activity.Elevated activity of beta-G in blood serum can be detected in patientswith early hepatic carcinoma; Elevated activity of beta-G in blood serumand CSF in patients with cerebral tumor can also be detected. Inaddition, some microorganism such as E. coli also has elevatedβ-glucuronidase activity, which can be used for detection of thesemicroorganisms in a sample, e.g., E. coli contamination in food. Thegeneral protocols of using these substrates for detection ofbeta-glucuronidase activity are similar to other enzyme detectiondescribed above and can be readily adopted by a skilled in the art.

The current invention also relates to novel chemiluminescenthydroxyproline aminopeptidase substrates that can be used to detecthydroxyproline aminopeptidase, which can cleave the substrates andrelease the hydroxyproline and the chemiluminescent moiety such asfirefly luciferin or 1,2-dioxetane derivatives, which enablesluminescence in their free form. Examples of these substrates are shownin FIG. 21. The free firefly luciferin can be quantified in abiochemiluminescence that uses firefly luciferase and ATP. Appropriateprotocols are similar to other enzyme detection described above and canbe readily adopted by a skilled in the art. Other substrates such ashydroxyproline-1,2-dioxetane derivative andhydroxyproline-coelenterazine (or its derivatives) (similar to thesubstrates used in proline aminopeptidase assay described above but theproline moiety is replaced by a hydroxyprolyl group) are also suitablefor detection of hydroxyproline aminopeptidase activity in achemiluminescent assay as long as the released chemiluminescent moietycan enable the production of light signal. Preferably the —OH on theproline is a trans-hydroxy group.

In one example, during the assay, the aminopeptidase cleaves thesynthetic substrate and releases the free 6-amino firefly luciferin,which can be quantified in a biochemiluminescent assay that uses fireflyluciferase and ATP. In yet another example, the 1,2-dioxetane substrateis used for chemiluminescent hydroxyproline aminopeptidase activitydetection. The assay protocol can be adopted from that which isdescribed in the proline aminopeptidase assay.

Hydroxyproline aminopeptidase assay can also be used to detect Neisseriagonorrhoeae. For example, the vaginal samples from neisseria gonorrhoeaepatients show high level of hydroxyproline aminopeptidase activity.Therefore, the substrates described in the current invention can be usedfor diagnosis of neisseria gonorrhoeae infection.

The current invention discloses a series of chemiluminescent substratesfor different enzymes. These substrates have the following formulas:

Wherein:

R_(L) is H or alkyl group of 1-20 carbon atoms such as methyl group; Tis a substituted or unsubstituted polycycloalkyl group bonded to the4-membered ring portion of the dioxetane by a Spiro linkage; X is anaryl or heteroaryl moiety of 6-30 carbon atoms which induceschemiluminescent decomposition of the 1,2-dioxetane upon enzymaticcleavage of moiety Rs; R is an alkyl, aryl, aralkyl or cycloalkyl of1-20 carbon atoms. Examples of R, X and T are described in U.S. Pat. No.6,555,698. The specific substitution group in the substrate for thecorresponding enzyme is shown in the table below:

Substitution symbol Substitution group Substrate for enzyme Rs-alpha-L-arabinopyranoside alpha-L-Arabinosidase Rs -beta-D-cellobiosidebeta-Cellobiosidase Rs -alpha-L-fucopyranoside alpha-L-Fucosidase(Describedin the above AFU assay) Rs -beta-D-fucopyranosidebeta-D-fucosidase Rs -beta-L-fucopyranoside beta-L-Fucosidase Rs-N-acetyl-alpha-D-galactosaminide alpha-Galactosaminidase Rs-N-acetyl-beta-D-galactosaminide beta-Galactosaminidase (Described inthe above Candida albicans test) Rs -alpha-D-galactopyranosidealpha-Galactosidase Rs -beta-D-galactopyranoside beta-Galactosidase Rs-N-acetyl-alpha-D-glucosaminide alpha-Glucosaminidase Rs-N-acetyl-beta-D-glucosaminide beta-Glucosaminidase (Described in theabove NAGase test) Rs -alpha-D-glucopyranoside alpha-Glucosidase Rs-beta-D-glucopyranoside beta-Glucosidase Rs -beta-D-glucuronic acidbeta-Glucuronidase (Described in the above beta- Glucuronidase test) Rs-beta-D-lactopyranoside beta-Lactosidase Rs -beta-D-maltopyranosidealpha-Maltosidase Rs -alpha-D-mannopyranoside alpha-Mannosidase Rs-beta-D-mannopyranoside beta-Mannosidase Rs -beta-D-xylopyranosidebeta-Xylosidase

As described above, the general principle for detecting these enzymeactivities using these listed chemiluminescent substrates involve twosteps. The first step is the enzyme cleaving the substrate and releasingthe chemiluminescent molecule. The second step is the chemiluminescentmolecule emitting light under suitable condition (e.g. high pH orcatalyzed by luciferase). In the first step, the ingredients in thereagent mix and the buffer need to be formulated to allow the targetenzyme cleaving the substrate (e.g. suitable pH and the presence ofcertain ion). These formulations are well known for the skilled in theart. Sometimes the condition suitable for the first step is alsosuitable for the chemiluminescent molecule emitting light. Therefore thetwo steps can be combined. Sometimes the condition suitable for thefirst step is not suitable for the chemiluminescent molecule emittinglight, therefore additional reagent and/or buffer need to be added afterthe cleavage of the substrate to satisfy the condition for thechemiluminescent reaction.

The present invention further provides methods and compositions forfirefly luciferase (or other luciferase utilizing firefly luciferin andATP, e.g. click beetle luciferase) based homogeneous enzyme channelingluminescent assays for analyte detection. Analyte is molecule orcomposition to be measured, which may be a ligand, small molecule, largemolecule, protein, enzyme, peptide, nucleic acid and etc.

The invention utilizes the enzyme channeling effect to detect theanalyte. Enzyme channeling effect utilizes two enzymes, which arerelated by the product of one being the substrate of the other.Therefore, when the two enzymes are close to each other a greaterturnover would be expected of the product of the first enzyme in theseries by the second enzyme in the series. Therefore, there will be atleast one compound (substrate) as part of a signal producing systemwhich is capable of being modified by a first enzyme to produce aproduct which will be modified by a second enzyme to produce a secondproduct which, directly or indirectly provides a detectable signal.

Current invention utilizes the enzyme channeling phenomenon to detectthe analyte. An enzyme channeling system is composed of two enzymes, inwhich the first enzyme acts on a substrate and produces a product thatis the substrate of the second enzyme. Enzymatic action of the secondenzyme produces a signal such as light signal that can be detecteddirectly or indirectly.

It is known to the skilled in the art that in the enzyme channelingsystem as described above, physical proximity of the two enzymes affectsthe signal output. The closer the two enzymes are, the stronger signalthere is. This phenomenon can be used to detect direct or indirectinteraction or binding of the two enzymes in the system. The interactionmay be a receptor-ligand interaction or its variations, anantibody-antigen interaction or its variations, a nucleic acidcomplementation based interaction or its variations, an aptamer basedinteraction or its variations, or the likes, including those that haveyet been discovered. A skilled in the art is able to devise a suitableinteraction for detecting a specific analyte.

One example of the enzyme channeling based detection system is bestunderstood by referring the example depicted in the FIG. 22. The firstenzyme 2 is coupled to the first receptor 4 through a chemical bond 3whereas the second enzyme 8 is coupled to the second receptor 6 via achemical bond 7. In the presence of a ligand/analyte 5 that has distinctbinding sites for the first and second receptors, the first and secondenzymes are brought together through the interaction of receptors andligand, thus forming an enzyme channel. The action of the first enzyme 2on the first substrate 1 (S1) produces the first product 9 (P1), whichis the substrate for the second enzyme 8. The second enzyme 8 convertsthe first product 9 (P1) to a detectable product 10 (P2). In thissystem, presence of an analyte results in higher signal. Thus, increasedsignal intensity indicates the presence of a specific analyte.

It is understood that the enzyme channels or the construction of thesechannels may vary from what is depicted in FIG. 22. For example, thefirst enzyme 2 and first receptor 4 can be conjugated to solid phasesupport such as micro- or nanoparticle. Similarly, the second enzyme 8and second receptor 6 can also be conjugated to solid phase support suchas micro- or nanoparticle. Because of the binding of a single analyte(ligand 5) can result in indirect interaction of multiple enzymemolecules, amplification can be achieved.

A number of techniques known to the skilled in the art can be used tocouple the enzymes to the ligands or receptor or the likes. For example,techniques are available for directly coupling a specific antibody withan enzyme such as alkaline phosphatase. The coupling can be done via acarrier system as well, e.g. a linker molecule, a polymer, a protein, asolid phase matrix such as a microsphere or nanoshpere. One example isthat both the second ligand/receptor and firefly luciferase are coupledwith a biotin group. The biotinylated ligand and firefly luciferase arethen used to coat an avidin coated microparticle or nanoparticle,resulting a microparticle or nanoparticle coated with both theligand/receptor and luciferase. The coupling/labeling techniques anddifferent labeling formats are well known to the skilled in the art andcan be found in varieties of publications.

In the enzyme channeling system described above, background signal maystill be produced even in the absence of an analyte (ligand 5), albeit,with weaker signal. However, a number of methods can be used to reducethis background signal. For example, a monoclonal antibody thatspecifically binds to the first product 9 (P1) can be used to sequesterthe first product (P1). This sequestering antibody competes with thesecond enzyme 8 for the first product 9.

However, presence of an enzyme channel, i.e., presence of ligand 5 (theanalyte) favors the first product to be channeled to the second enzyme.Alternatively, a degrading enzyme that competes with the second enzyme 8for the first product 9 but does not generate detectable signal can alsobe used to reduce the background.

It is understood that conditions need to be optimized to achieve thebest signal to noise ratio for an assay. For example, the optimalconcentrations of the enzyme conjugates, sequestering antibody, andsubstrates etc may be experimentally determined using methods known tothe skilled in the art.

The current invention is based on the use of firefly luciferinluminescent systems. In some embodiments, the two enzymes utilized forenzyme channeling are firefly luciferin producing enzyme and fireflyluciferase or other luciferase utilizing firefly luciferin such as clickbeetle luciferase. In other embodiments, those two enzyme utilized areATP producing enzyme and firefly luciferase or other luciferaseutilizing ATP such as click beetle luciferase.

When firefly luciferin producing enzyme is used, substrate that can beused by the firefly luciferin-producing enzyme to produce fireflyluciferin is also needed in the assay. Examples of thesesubstrate-firefly luciferin producing enzyme are listed below: fireflyluciferin methyl ester and carboxylic esterase, firefly luciferinO-sulfate and arylsulfatase, firefly luciferin O-phosphate and alkalinephosphatase, firefly luciferyl-L-N alpha-arginine or fireflyluciferyl-L-phenylalanine and carboxypeptidases A, B and N,firefly-luciferin-O-beta-galactoside and beta-galactosidase, otherexamples can be found in U.S. Pat. No. 5,098,828, US Patent 20070015790.The enzyme and its corresponding substrate described in the early partof this invention such as N-acetylneuraminic acid-firefly luciferinconjugate and sialidase, L-prolyl-6-amino firefly luciferin and prolineaminopeptidase are also suitable for this method. The generalrequirement for these substrate-enzyme pair is that the enzyme should beable to convert the corresponding substrate and generate fireflyluciferin or its analogue that can produce light signal by fireflyluciferase while the unconverted substrate does not luminesce byluciferase or only luminesce very weakly. In certain embodiments, aluciferin sequestering antibody is used in the system to reduce thebackground.

When ATP producing enzyme is used, free firefly luciferin or itsanalogue (e.g. firefly 6-amino luciferin) that can luminesce by fireflyluciferase or other luciferase utilizing firefly luciferin such as clickbeetle luciferase is needed in the system instead of the substrate thatcan be converted to firefly luciferin such as those described above. Inthis system, an ATP producing enzyme is used as the first enzyme toproduce ATP that enables luciferase-based chemiluminescence reaction. Inthis enzyme channeling system, free firefly luciferin or its analogue(e.g. firefly 6-amino luciferin) is still used in the reaction. Anyenzyme that can produce ATP can be used in the invention such asnucleoside diphosphate kinase, phosphoglycerate kinase, pyruvate kinase,sulfurylase, acetate kinase and the corresponding substrates that canproduce ATP such as ADP are also need to be provided. In another word,any enzyme that can produce ATP can be used as the first enzyme in thepresent invention. Examples of these enzymes include, but are notlimited to, phosphoglycerate kinase and pyruvate kinase. Appropriatesubstrates and conditions need to be provided in order for the enzyme toproduce ATP. For example, 1,3-biphosphoglycerate and ADP must be presentin order for phosphoglycerate kinase to convert ADP to ATP. Similarly,phosphoenolpyruvate and ADP must be provided in order for pyruvatekinase to produce ATP, which is subsequently used in the fireflyluciferase-catalyzed chemiluminescent reaction. In another example,sulfate adenylyltransferase (ATPS) is used, which converts APS(adenosine 5′-phosphosulfate) to ATP in the presence of PPi, forgenerating ATP.

In order to detect a specific analyte, two affinity ligands need to beprovided, the first ligand can bind with one part/area of the analyteand the second ligand can bind with another part/area of the analyte andtherefore can form a sandwich type structure similar to those in theclassical ELISA type assay. Suitable ligands include antibody, nucleicacid, small molecule, protein, aptamer, receptor and etc. These types ofligands-analyte complex are very common in modern assay and are wellknown to the skilled in the art. In the current invention, the firstligand (or ligands, e.g. multiple copies of ligand having same bindingmode) is coupled (sometimes called labeled) with firefly luciferinproducing enzyme/enzymes (or ATP producing enzyme/enzymes) and thesecond ligand (or ligands) is coupled with fireflyluciferase/luciferases. The coupling can be done via a carrier system,e.g. a linker molecule, a polymer, a protein, a solid phase matrix suchas a microsphere. One example is that both the first ligand and fireflyluciferase have a biotin group and upon being mixed with an avidincoated solid phase support such as micro particle the first ligand andfirefly luciferase will be immobilized on the solid phase supportgenerating the ligand labeled firefly luciferin. In this case, it ispossible that each particle carry multiple copies of the first ligandmolecule and firefly luciferase. The coupling/labeling/coatingtechniques and different labeling formats are well known to the skilledin the art and can be easily found in varieties of publications.

For example, in an assay to detect a specific protein A, two monoclonalantibodies against different regions of protein A is used. The firstantibody is labeled with phosphatase, the second one is co immobilizedtogether with firefly luciferase on the solid phase support (e.g.Sepharose beads). Alternatively, the second antibody can be directlylabeled with firefly luciferase. When these is protein A in the sample,upon incubation, a sandwich structure will form which can be describedas first antibody-phosphatase-protein A-second antibody-fireflyluciferase and therefore the firefly luciferase is very close to thephosphatase. The corresponding substrate firefly luciferin O-phosphatecan be added before or during or after the incubation. The phosphatasewill cleave the phosphate group and release the free firefly luciferinand the released firefly luciferin will be consumed preferably by thenearby firefly luciferase and generate light signal which can be in turnmeasured with suitable means such as a luminometer. The assay solutionis formulated to contain all the reagents and condition such as ATP andsuitable pH necessary for the activity for both enzymes. No free fireflyluciferin is added. This assay can be performed directly in the samplesolution. The protocol is very simple and no separation step is requiredto remove the excess unbound labeled antibodies. The assay protocol islimited to the addition of sample, substrate, and other reagents. Thelight signal generated is related to the amount of sandwich structureformed and therefore related to the amount of analyte in the assay.Because the unbound phosphatase will also produce free firefly luciferinthat can generate light signal when it is consumed by bound or unboundfirefly luciferase and therefore may cause background luminescencesignal, an antibody that can specifically bind with free fireflyluciferin and block its ability to luminesce can be added to the assay,however, this antibody should not interfere the substrate beingconverted to free firefly luciferin by the firefly luciferin producingenzyme. The amount of the antibody added can be determinedexperimentally to reach the optimal signal/noise ratio. This antibodyworks as a scavenger to neutralize the background producing fireflyluciferin since the firefly luciferin generated within the sandwichcomplex will less likely been blocked by the antibody.

When ATP producing enzyme is used for the above assay, no fireflyluciferin producing enzyme (e.g. phosphatase) labeled antibody isrequired. The two antibodies would be labeled with ATP producing enzymeand firefly luciferase respectively. Free firefly luciferin is addedeither before or during or after the sandwich structure formed (e.g. theincubation). Substrate for ATP producing enzyme such as ADP is addedinstead of the firefly luciferin O-phosphate either before or during orafter the incubation. The formulation and protocol need to providecondition for both ATP producing enzyme and firefly luciferase'sactivity. In order to decrease the background noise, instead of fireflyluciferin specific antibody, an ATP eliminating enzyme such as ATPhydrolysase can be added to degrade the ATP generated not within thesandwich complex, which may cause background light signal. In thisassay, no free ATP needs to be added. The light generated solely dependson the ATP produced by the ATP producing enzyme.

There are many scientific publications and patents described ATPproducing enzyme suitable for the current invention. For example, U.S.Pat. No. 5,246,834, which is cited here solely for reference, describedan EIA method using an ATP-generating enzyme as a labeling enzyme. TheATP generated by the enzyme is detected in a bioluminescence assay thatuses firefly luciferase. The emitted light signal is proportional to theamounts of the analyte in the sample. In this patent, acetate kinase isa preferred enzyme for generating ATP. The enzyme, reagent and protocolused for ATP generating in U.S. Pat. No. 5,246,834 can be readilyadopted by the skilled in the art for the homogenous assay described inthe present invention.

There are also many scientific publications and patents described ATPeliminating enzyme suitable for the current invention. For example, U.S.Pat. No. 5,891,702, which is cited here solely for reference, describeda process for eliminating effectively ATP in a sample, using adenosinephosphate deaminase alone or in combination with at least one enzymeselected from the group consisting of apyrase, alkaline phosphatase,acid phosphatase, glycerokinase, hexokinase and adenosinetriphosphatase. The enzymes, reagents and protocol used for ATPeliminating in U.S. Pat. No. 5,891,702 can be readily adopted by theskilled in the art for the homogenous assay described in the currentinvention.

The assay for other analyte such as small molecule and nucleic acid canalso be performed based on the above principle. The reagents andprotocol can be easily adopted form the above case by a skilled in theart. Alternatively, competitive binding assays can also be used based onthe same mechanism described above. The principle and protocol of thecompetitive assay is well known for the skilled in the art. For example,in an assay to detect a specific protein A or small molecule A, onemonoclonal antibody against A is used. The antibody is labeled withphosphatase and A is labeled with firefly luciferase (or A is labeledwith phosphatase and the antibody is labeled with firefly luciferase).The assay solution contains preformed sandwich structureantibody-phosphatase-A-firefly luciferase and therefore the fireflyluciferase is very close to the phosphatase. After it is incubated withthe sample, if there is A in the sample, some of the aboveantibody-antigen complex will disassociate proportional to the amount offree A in the sample because antibody-phosphatase-A will formcompetitively. The corresponding substrate firefly luciferin O-phosphatecan be added before or during or after the incubation. The phosphatasewill cleave the phosphate group and release the free firefly luciferinand the released firefly luciferin will be consumed preferably by thenearby firefly luciferase and generate light signal which can be in turnmeasured with suitable means such as a luminometer. Now the intensity oflight produced is proportional to the amount ofantibody-phosphatase-A-firefly luciferase complex which in turn isdetermined by the amount of free A in the sample. The more A in thesample, the lower the light produced.

EXAMPLE 1 Detection of Influenza Viral Neruaminidase

In this example, the influenza test kit contains two key components:conjugate mix and detection mix.

Reagent Compositions

Conjugate Mix (lyophilization is preferred) MES, pH 6.5 32.5 mM CaCl₂ 4mM BSA 1 mg/mL Triton X-100 0.5%   Mannitol 4% Sucrose 1% Conjugate 100μg/mL Detection Mix (lyophilization is preferred) Trizma, pH 7.8 100 mMMgSO₄ 15 mM BSA 1 mg/mL ATP, Na Salt 12 mM NP₄₀ or Equivalent 0.1%   DTT10 mM Co-enzyme A 1 mM EDTA, Na Salt 2 mM Mannitol 4% Sucrose 1% FireflyLuciferase 20 μg/mL

Assay Protocol

In this example, the influenza virus detection assay comprisesessentially two steps: 1) cleavage of sialic acid-firefly luciferinconjugate with influenza neuraminidase, and 2) detection of releasedfirefly luciferin. Specifically one can use the following basicprotocol:

1. Mix the sample containing flu virus with 100 μL conjugate solution,which will lyse the virus because of the presence of Triton X-100.

2. Incubate at room temperature for 10-15 minutes.

3. Transfer the solution to 100 μL firefly luciferase reaction solutionpre-loaded into a detection tube, or a lyophilized detection mix in adetection tube.

4. Place the detection tube into a luminomter and record the lightsignal (relative light unit) for an appropriate period of time (e.g., 30seconds).

Note that firefly luciferase mediated biochemiluminescence reaction isof a glow light type, which stably emits light for at least 5 minutes.Therefore, there is no need to use a luminometer with an automatedinjector. Click beetle luciferase can also be used instead of thefirefly luciferase.

EXAMPLE 2 Detection of Influenza Viral Neuraminidase in a One Step RealTime Detection Format

In brief, influenza virus collected in the throat nasal swab is lysed ina virus lysis buffer (PBS+1% Triton X 100). A portion of the lysisbuffer (200 μL) is then added to a pre-mix containing all necessaryreagents, followed by incubation for 10-15 minutes at room temperature.Presence of influenza virus, hence the viral neuraminidase, results inthe 995 cleavage of a substrate, which enables the generation of visiblelight signal that is detected with a portable luminometer. Click beetleluciferase can also be used instead of the firefly luciferase.

Detection Mix (Lyophilized Form Preferred)

Imidazole, pH 7.0-7.2 50 mM BSA 1 mg/mL ATP, Na Salt 12 mM DTT 10 mMCo-enzyme A 1 mM or 10 mg/mL MgSO4 15 mM CaCl2 4 mM Mannitol 4% Sucrose1% Sodium Azide 0.05%   Firefly Luciferase 20 μg/mL Substrate 20 μg/mL(or as determined by QC test)

Assay Protocol

Step 1—Sample preparation:

Place the throat swab into the Virus Lysis Buffer tube,

Roll the swab at least three times while against the bottom and side ofthe tube,

Wring out the swab by squeezing the tube wall against the swab andcarefully pulling out the swab from the tube.

Discard the swab in a biohazard container.

Step 2—Transfer 200 μL of the sample prepared in Step 1 to a DetectionMix test tube. Cap the vial. Gently swirl the tube until all of thelyophilized powder is wet. Particulate materials may initially bepresent in the mix, which does not interfere with the detection.

Step 3—Incubate at room temperature (20-30° C.) for 15 minutes.

Step 4—Place the test tube in the luminometer and press the startbutton. Record and print the test results.

EXAMPLE 3 Use of Antibodies for Inhibiting Non-Specific NeuraminidaseActivity in an Influenza Viral Neuraminidase Assay

The commonly used clinical samples for influenza detection are throatand nasal swabs. Some bacterial species that are found in nasal or oralcan also secret neuraminidase. These bacterial species includeStreptococcus pneumoniae and Actinomyces viscosus. In this example,monoclonal or polyclonal antibodies specific for the neuraminidases forthese bacterial species are added to the conjugate mix in Example 1 orthe lysis buffer in Example 2. Bacterial neuraminidase in the sample, ifany, is blocked by the antibodies thereby reducing the background due tonon-specific bacterial neuraminidase.

EXAMPLE 4 Diagnosis of Bacterial Vaginosis Using an N-AcetylneuraminicAcid-Firefly Luciferin as the Chemiluminescent Substrate

In this example, an N-acetylneuraminic acid-firefly luciferin conjugateis used as the chemiluminescent substrate for detection of bacterialvaginosis. A detection mix described in Example 2 can be used for thispurpose. However, it is understood that the detection mix can beoptimized for bacterial vaginosis detection since sialidase activity ina vaginal sample may be considerably higher than that for a sample forinfluenza detection.

The detection mix solution described in Example 2 is used to demonstratethe detection of bacterial vaginosis. The Osom BV blue positive controland negative control (from Genezyme Diagnostics), which containdifferent levels of bacterial sialidase used for bacterial vaginosisdiagnosis, are used to demonstrate the use of the detection mix forbacterial vaginosis detection. 40 microliters of the control sample wasmixed with 50 microliters of detection mix described in Example 2 inroom temperature and immediately placed in a luminometer. Thechemiluminescence started instantly for the positive control. Thechemiluminescence signal (relative light unit) from the positive controlwas 300 times higher than the negative sample, demonstrating that thedetection mix could well separate the positive samples from the negativeones. The assay is highly sensitive and quantitative. In addition, theassay requires no incubation, which greatly reduces the assay time.

For detection that uses a patient sample, i.e., vaginal swab, thevaginal swab can be first rinsed in a sample buffer, e.g., 1 mL of 1×PBS buffer, which or a portion of which is mixed with the detection mixin solution form or, preferably, in lyophilized form in a test tube. Thetest tube is placed in a luminometer for detection. It is understoodthat a cutoff value for the diagnosis in terms of light signal intensity(RLU) needs to be established by testing a large number of negative andpositive samples, preferably more than 100 positive samples and 100negative samples.

EXAMPLE 5 Diagnosis of Bacterial Vaginosis Using an N-AcetylneuraminicAcid-Dioxetane as the Chemiluminescent Substrate

In addition to the N-acetylneuraminic acid-firefly luciferin conjugatesdescribed in Example 4, other chemiluminescent conjugates can also beused for bacterial vaginosis detection. In this example, 3 mg of adioxetane conjugate depicted in FIG. 7 or 8 is dissolved in 1 mL of 0.5M sodium acetate buffer (pH 7.6). 10 uL of this detection solution ismixed in a test tube with 200 microliters of vaginal fluid sample, whichis prepared by rinsing a vaginal swab in 1 mL 0.1 M PBS buffer (pH 7.6).The test tube is placed in a luminometer, which reads and integrates thesignal for 10 seconds to 3 minutes. Again, a cutoff value in terms oflight signal intensity (RLU) needs to be established by testing a largenumber of negative and positive samples, preferably more than 100positive samples and 100 negative samples.

The dioxetane conjugate can be lyophilized for long-term storage, inwhich case the vaginal fluid sample can be directly added to thelyophilized dioxetane conjugate mix followed by detection using aluminometer. The test can also follow the protocol described inAnalytical Biochemistry 280, 291-300 (2000) in a two steps manner:first, the substrate is incubated with the sample for 5-10 minutes atlow pH (e.g. pH 5˜6), then mixed with the high pH triggering reagent(e,g, by adding 100 uL of the Sapphire enhancer, pH10.5) to initiate thechemiluminescence for reading. The one step method is preferred becauseit simplifies the assay. Because at low pH, the dioxetane has lowchemiluminescence, the preferred pH for the one step assay is between7˜8.5.

EXAMPLE 6 Diagnosis of Candida albicans Infection Using ChemiluminescentSubstrates

In this example, 1,2 dioxetane-N-acetyl-beta-D-galactosaminide substrateis used for detection of the beta-galactosaminidase activity whereasL-propyl-1,2-dioxetane derivative substrate is used for detection ofaminopeptidase activity.

The clinical sample (e.g. vaginal fluids) is suspended in 500microliters of 0.1 M PBS buffer (pH 7.0). 250 microliters of this sampleis mixed with 10 microliters of beta-galactosaminidase detection mix (3mg of dioxetane substrate in 1 mL of 0.5 M sodium acetate buffer at a pHof about 6.5) in a vial, incubated for 5-10 minutes at 30 degree C.,mixed with 100 microliters of trigger reagent (Sapphire enhancer,pH10.5) and measured for the light signal using a luminometer.Interpretation of the test result (positive or negative forbeta-galactosaminidase) is based on the established cutoff value.

The remaining 250 microliters of sample is mixed with 10 microliters ofL-proline aminopeptidase detection mix (3 mg of dioxetane substratedissolved in 1 mL of 0.5 M sodium acetate buffer at a pH of about 8.0)in another vial, incubated for 5-10 minutes 30 degree C., mixed with 100microliters of trigger reagent (Sapphire enhancer, pH10.5) and thenmeasured for the light signal using a luminometer. Interpretation of thetest result (positive or negative for aminopeptidase) is based on theestablished cutoff value. Infection with Candida albicans is indicatedwhen both the activities for both enzymes exceed the cutoff values.

EXAMPLE 7 Detection of an Antigen Using a Firefly Luciferase BasedEnzyme Channeling System

In this example, the enzyme channeling based assay is used to detect aspecific antigen, antigen A, for which there are two monoclonalantibodies against different regions of the antigen. The first antibodyis coupled with alkaline phosphatase whereas the second one is coupledwith firefly luciferase. In the enzyme channeling system, theappropriate corresponding substrate is firefly luciferin O-phosphate.Therefore, a detection mix contains three key components (firstantibody-alkaline phosphatase conjugate, second antibody-fireflyluciferase conjugate, and firefly luciferin O-phosphate) and otherappropriate conditions that enable the reactions to occur. Preferably,the detection mix is lyophilized to maintain long-term stability. Inpreferred embodiments, the detection mix also contains an antibody thatbinds to free firefly luciferin but not firefly luciferin O-phosphate,which reduces the background.

An example of the detection procedure is as follows: a sample from anappropriate source is diluted in a buffer (e.g., 1 mL of 1× PBS buffer),which is then added to the lyophilized detection mix in a detectiontube. After thorough mixing, the detection tube is placed in aluminometer for detection. In the presence of antigen A, a sandwichstructure similar to depicted in FIG. 16, thereby causing the fireflyluciferase to be closed to the phosphatase. Close proximity of the twoenzymes results in more efficient signal production, e.g., enhancedsignal output, in comparison with the control that contains no antigen A(the analyte). This is because close proximity of the two enzyme causesthe released free firefly luciferin to be immediately consumed byfirefly luciferase to produce stronger light signal, which indicates thepresence of the analyte. In particular, when there is a fireflyluciferin-sequestering entity (e.g., firefly luciferin-binding protein)in the detection mix, the background is much lower, resulting in anenhanced signal to noise ratio.

In certain embodiments, the substrate (firefly luciferin O-phosphate) isnot contained in the detection mix. In stead, the substrate is containedin the sample dilution buffer. This will prevent the substrate frombeing degraded during manufacturing, which may take several hours fromformulation, dispensing to individual detection tube to lyophilization.In other embodiments, the firefly luciferin-binding antibody is alsoadded to the sample dilution buffer along with the substrate, which mayreduce the free firefly luciferin produced in the dilution buffer due toauto-cleavage of the substrate.

It is understood that concentration of various components in thedetection mix (e.g., antibody-enzyme conjugates, fireflyluciferin-binding antibody etc) need to be optimized, which can beperformed using protocols well known to the skilled in the art.

EXAMPLE 8 Detection of an Antigen Using a Firefly Luciferase BasedEnzyme Channeling System

In this example, ATP is used as a limiting factor for detection in thefirefly luciferase based channeling system. When ATP producing enzyme isused in the system, no firefly luciferin-producing enzyme (e.g.phosphatase) labeled antibody is required. The first antibody is coupledto an ATP producing enzyme whereas the second antibody is coupled withthe firefly luciferase. Free firefly luciferin is added in the detectionmix in appropriate concentration.

In this example, the ATP-producing enzyme is pyruvate kinase, whichconverts ADP to ATP in the presence of phosphoenolpyruvate underappropriate conditions (e.g., pH, salt composition). Therefore, in thissystem, the substrates are ADT and phosphoenolpyruvate instead of thefirefly luciferin O-phosphate described in Example 7. In order todecrease the background noise, an ATP degrading enzyme such as ATPhydrolysase can be added to the detection mix to degrade the ATP notproduced in the two-enzyme complex. This is in contrast with the exampledescribed in Example 7, where firefly luciferin binding antibody is usedto reduce the background. Essentially, the ATP degrading enzyme competeswith firefly luciferase, the latter producing the light signal in achemiluminescence reaction. When firefly luciferase forms a complex withthe ATP-producing enzyme in the two enzyme channeling system, fireflyluciferase is advantageous in competing for ATP because of its physicalproximity to the ATP producing enzyme.

Detection of ATP with firefly luciferase is well established in the artfor detection of microorganisms or contaminating biological tissues. Theconditions are similar to what are described in Examples 1 and 2.

The detection mix can be formulated to contain all necessary reagentsand chemicals for both enzymatic reactions except for ADP (one of thesubstrates) and ATP degrading enzyme, both of which are preferably, butnot necessarily, contained in the sample dilution buffer (e.g., 1× PBSbuffer).

For detection of antigen A, the detection mix contains the firstantibody-pyruvate kinase conjugate, the second antibody-fireflyluciferase conjugate, phosphoenolpyruvate, firefly luciferin,appropriate amounts of salts and pH conditions, 1% sucrose, and 8%mannitol. This detection mix is preferably lyophilized for long-termstorage of the detection mix.

The sample dilution buffer may contain 1× PBS, appropriate amounts ofADP and appropriate amounts of ATP hydrolyase. Presence of ATPhydrolyase in the sample dilution buffer can also eliminate ATP that islikely present in a biological sample and in ADP reagent.

An example of the detection procedure is as follows: a sample from anappropriate source is diluted in the sample dilution buffer describedabove (e.g., 1:100 dilution). Incubation may be necessary to allow forthe ATP in the sample to be degraded by ATP hydrolyase in the buffer. 1mL of the diluted sample is then added to the lyophilized detection mixin a detection tube. After thorough mixing, the detection tube is placedin a luminometer for detection. In the presence of antigen A, a sandwichstructure similar to that depicted in FIG. 22 is formed, thereby causingthe firefly luciferase to be in close proximity to the pyruvate kinase.Being in close proximity to pyruvate kinase, which converts ADP to ATP,firefly luciferase can more effectively compete with ATP hydrolyase forATP, thereby resulting in enhanced signal output when compared to thecontrol that contains no antigen A (the analyte). Therefore, increasedsignal indicates the presence of the analyte.

EXAMPLE 9 Detection of Nucleic Acid Sequences, Peptides, and SmallMolecules Using a Firefly Luciferase Based Enzyme Channeling System

Detection of other analyte types such as peptides, small molecules andnucleic acids can also be performed using an enzyme channeling system orits variations described in the present invention. Hybridization ofnucleic acid sequences can also be detected since they can form complexthat brings the two-enzymes physically close together. In a system fordetecting a nucleic acid sequence with two hybridization domains, thetwo enzymes can be coupled with two distinct probes, one for eachdomain. Presence of the target nucleic acid sequence will cause the twoenzymes to be hybridized to the same nucleic acid sequence and thereforebe in close proximity.

Sometimes small molecules that have only one ligand-binding domain canalso be detected with the enzyme channeling system described in thecurrent invention. Detection of small molecules with only oneligand-binding domain, however, requires a competition-based assay. Forexample, one of the enzymes (e.g., pyruvate kinase) is coupled with anantibody specific for the small molecule whereas the luciferase iscoupled with the small molecule itself. When these two conjugates aremixed together in a detection mix along with substrates and underappropriate conditions, a signal is generated. When a sample containingthe small molecule is added, the signal is reduced because the smallmolecule in the sample competes with those coupled to firefly luciferasefor the antibody coupled to the other enzyme. When ATP producing enzymeis used in the system, ATP hyrolyase (or other ATPases) can be used toimprove the signal to noise ratio. In this case, a reduction of thesignal indicates the presence of analyte in the sample.

One detailed example is given below: the assay is to detect the peptidehuman chronic gonadotropin (hCG) in the serum sample. The firstmonoclonal antibody for human chronic gonadotropin (hCG) is linked withbacterial sialidase, the second monoclonal antibody for hCG, whichrecognizes a different portion of the hCG molecule than that recognizedby the first monoclonal antibody is linked with firefly luciferase.

The detection solution contains the following:

Imidazole (pH 7.0) 50 mM BSA 1 mg/mL ATP, Na Salt 12 mM DTT 10 mMCo-enzyme A 1 mM or 10 mg/mL MgSO4 15 mM CaCl₂ 4 mM Mannitol 4% Sucrose1% Sodium Azide 0.05%   Firefly Luciferase-antibody conjugate 2 μg/mLSialidase-antibody conjugate 2 μg/mL

100 microliters of assay solution described above is mixed with100microliters of HCG containing sample and incubated at 25 degree C. for10 minutes. Next, 100 microliters of substrate (N-acetylneuraminicacid-firefly luciferin conjugate, 50 micrograms/mL) solution is added tothe reaction mix and placed in a luminometer for reading the lightsignal. The higher the light signal reading, the higher the HCGconcentration is in the sample. It is preferred that a background signalis established by testing a large number (e.g., 50 samples) and a linearcurve is established by testing serially diluted positive samples. It ispreferred that an antibody against firefly luciferin is included in thedetection mix to increase the signal to noise ratio.

Yet another more detailed example is given below: the assay is to detecta target nucleic acid sequence in a competition assay format. The targetsequence is CCCCCCCCCCCC. Streptavidin agarose (from Invitrogen, CatalogNumber SA100-04) is coated with biotin labeled firefly luciferase andbiotin labeled DNA sequence-biotin by incubating the agarose beads withthe biotinylated luciferase and DNA sequence. After washing to get ridof the excess firefly luciferase and G-biotin, CCCCCCCCC-alkalinephosphatase conjugate is incubated with the bead and the resultingbead-GGGGGGGGG-CCCCCCCCC-alkaline phosphatase complex is again washed toget rid of the excess CCCCCCCCC-alkaline phosphatase conjugate. Theresulting beads are called detection beads.

The assay formulation contains the following:

Imidazole (pH 8) 50 mM BSA 1 mg/mL ATP, Na Salt 12 mM DTT 10 mMCo-enzyme A 1 mM or 10 mg/mL MgSO4 15 mM CaCl₂ 4 mM Mannitol 4% Sucrose1% Sodium Azide 0.05%   Detection beads 2 μg/mL

100 microliters of assay solution described above is mixed with 100microliters of sample and incubated at 25 degree C. for 30 minutes. Next100 microliters of substrate (firefly luciferin O-phosphate, 50micrograms/mL) solution is added to the reaction mix and placed in aluminometer for reading the light signal. The lower the light signalreading, the higher the target nucleic acid concentration is in thesample. It is preferred that a background signal is established bytesting a large number (e.g., 50 samples) and a linear curve isestablished by testing serially diluted positive samples. It ispreferred that an antibody against firefly luciferase is included in thedetection mix to increase the signal to noise ratio.

EXAMPLE 10 Detection of Human IgG from HIV Positive Serum Using aFirefly Luciferase Based Enzyme Channeling System

The current example utilizes ATP producing enzyme-based enzymechanneling system. In the current example sulfate adenylyltransferase(ATPS) is used, which converts APS (adenosine 5′-phosphosulfate) to ATPin the presence of PPi, for generating ATP. The ATPS is fused with ZZdomain (ATPS-ZZ), which can bind with IgG. Another affinity ligand isgp160, which can bind with the HIV positive IgG. The ATP utilizingluciferase and one of the affinity ligand are co-immobilized on solidsupport. The firefly luciferase or click beetle luciferase isbiotinylated. The gp160 is also biotinylated. Avidin coated EIAstripwell plate is coated with these biotin labeled firefly luciferase(or click beetle luciferase) and gp160. Some of the suitable ratio ofcoated firefly luciferase and coated gp160 is between 10:1 to 1:2. Thecoating of biotin labeled protein to avidin plate is well known to theskilled in the art and the biotinylated firefly luciferase and gp160 arecommercially available. Each well is added with 50 ng of ATPS-ZZ in 150uL PBS buffer containing 0.1% BSA as well as different amount of IgG.Incubation is performed for 20 min under room temperature. Next 50 uLHBA buffer is added to each well and the light signal is collected witha luminometer for 2 min. HBA buffer contains 0.1M Tris-Acetate, pH 7.75;2 mM EDTA, 10 mM Mg Acetate; 0.1% BSA; 1 mM DTT; 100 ug/ml D-Luciferin;5 uM APS; 100 uM PPi; 0.25 mg/ml CoA and 0.4 mg/ml PVP. The reading ofthe well containing 10 ng of IgG is 5 times the reading of the wellcontaining no IgG. In order to decrease the back ground, apyrase (frompotato) can be added to the HBA buffer as ATP eliminating enzyme. One ofthe suitable concentration of apyrase in the final mix is 0.01˜0.5units/mL.

This assay can also be done without the need of solid phase support. Inone embodiment, gp160 and firefly luciferase are conjugated directlywithout being immobilized on solid support. 25 ng of gp160-fireflyluciferase conjugate and 50 ng of ATPS-ZZ in 200 uL PBS buffercontaining 0.1% BSA are incubated together with different amount of IgGfor 10 min. Next 50 uL HBA buffer is added and the light signal iscollected with a luminometer for 2 min for the detection. The apyrasecan also be added to reduce the background signal.

What is claimed is:
 1. A compound of formula I

where in T is a substituted or unsubstituted polycycloalkyl group bondedto the 4-membered ring portion of the dioxetane by a Spiro linkage; X isan aryl or heteroaryl moiety of 6-30 carbon atoms which induceschemiluminescent decomposition of the 1,2-dioxetane upon enzymaticcleavage of moiety Rs; R is an alkyl, aryl, aralkyl or cycloalkyl of1-20 carbon atoms; Rs is selected fromalpha-L-arabinopyranoside,beta-D-cellobioside,alpha-L-fucopyranoside,beta-D-fucopyranoside,beta-L-fucopyranoside,N-acetyl-alpha-D-galactosaminide,N-acetyl-beta-D-galactosaminide,alpha-D-galactopyranoside,beta-D-galactopyranoside,N-acetyl-alpha-D-glucosaminide,N-acetyl-beta-D-glucosaminide,alpha-D-glucopyranoside,beta-D-glucopyranoside,beta-D-glucuronicacid,beta-D-lactopyranoside,beta-D-maltopyranoside,alpha-D-mannopyranoside,beta-D-mannopyranoside andbeta-D-xylopyranoside.
 2. A method to detecting analyte in a sample,comprising: a) contacting said sample with a first ligand of the analytecoupled with ATP producing enzyme and a second ligand of the analytecoupled with luciferase, wherein said luciferase use ATP as substrate;and b) detecting the light generated from said luciferase.
 3. A kit fordetecting analyte in a sample, comprising a first ligand of the analytecoupled with ATP producing enzyme and a second ligand of the analytecoupled with luciferase.